"In situ"--measurements of protein contents in the brush border region along rat jejunal villi and their correlations with four enzyme activities.
In order to quantify the amount of protein in the small intestinal brush border region at different villus sites, cryostat sections of adult rat jejunum were stained with Naphthol Yellow S, Dinitrofluorobenzene and Coomassie Brilliant Blue and the dye deposits were evaluated cytophotometrically. Judged by the absorbance spectra in the tissue sections and the increase in absorbance as a function of the optical pathway (section thickness), Naphthol Yellow S proved to be the most suitable quantitative protein stain. By continuously measuring the absorbance of this dye at lambda 440 nm rectangular to the villus in the longitudinal axis of the enterocytes, a peak was registered in the brush border region which clearly could be differentiated from the apical cytoplasm. The amount of protein in the brush border region was determined at six different positions equally distributed along the villus. In parallel four brush border enzymes (neutral alpha and beta-glucosidase, unspecific alkaline phosphatase and dipeptidylpeptidase IV) were quantified by the same measuring technique in the Vmax-range of their substrate hydrolysis at equivalent villus positions. Their activities were correlated to the amount of protein. The absorbance data both for protein and for enzyme activities were significantly influenced by the villus position. They revealed an increasing gradient from the basal to the apical villus. In an additional analysis of the breadth of the dye deposits at the different measuring positions on the villus, it was shown that this parameter also ran parallel with the absorbance values.
D000469:Alkaline Phosphatase / Q000032:analysis*; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology*; D004139:Dinitrofluorobenzene; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000032:analysis*; D010450:Endopeptidases / Q000032:analysis*; D005260:Female; D005959:Glucosidases / Q000032:analysis*; D007583:Jejunum / Q000201:enzymology*; D008871:Microvilli / Q000201:enzymology*; D009282:Naphthalenesulfonates; D011506:Proteins / Q000032:analysis*; D051381:Rats; D012394:Rosaniline Dyes; D013053:Spectrophotometry; D014018:Tissue Distribution; D000520:alpha-Glucosidases / Q000032:analysis*; D001617:beta-Glucosidase / Q000032:analysis*
D009282:Naphthalenesulfonates; D011506:Proteins; D012394:Rosaniline Dyes; C009824:naphthol yellow; C024757:Coomassie brilliant blue R; D004139:Dinitrofluorobenzene; D000469:Alkaline Phosphatase; D005959:Glucosidases; D000520:alpha-Glucosidases; D001617:beta-Glucosidase; D010450:Endopeptidases; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases